The present invention relates to a novel microbe belonging to Serratia family, its isolation method and the preparation method using it. More particularly, the present invention relates to a microorganism of Serratia family producing laccase, lignin peroxidase and Mn-dependent-peroxidase which are the kinds of lignin lyases, and a method for separating the microbe from the gut of insect, as well as a method for preparing laccase, lignin peroxidase and Mn-dependent-peroxidase.
Lignin is the second most abundant material on earth after cellulose, and it is a kind of macromolecular compound existing in middle lamella and first cell wall of plant (Munir, T., et al., Enzyme and Microbial Technol., 1999, 25, 38–47). Lignin takes 15–35% of a total dried wood weight and exists in middle lamella between cell membrane as a high-molecular aromatic condensation compound while some of lignin exists in cell membrane. Lignin has various structures, but generally it has the structure of propylbenzene derivatives having substituent such as methoxyl or hydroxyl group in aromatic ring. Due to its complicated structure and stability, lignin has significant resistance against biological attacks. None of organism, though, using this as only carbon. source for their growth has been known(Higuchi, Experientia, 1982, 38, 159–166; Janshekar, H. and Feichter, A., Advances in Biochemical Engineering/Biotechnol., 1983, 27, 119–178).
Precursors composing lignin are ρ-coumaric alcohol, coniferyl alcohol, sinapyl alcohol and cinnamyl alcohol. The composition ratio of these precursors varies with the type, age and tissue of a plant. When the above mentioned precursors are going under oxidation by peroxidase existing in living matter, free radicals are formed as an intermediate product. These formed free radicals respond each other to be a pair and polymerized resulting in a form of macromolecule having phenyl-propane as a basic unit. That macromolecule is lignin (Hilden, L., et al., FEBS Letters, 2000, 477, 79–83; Leonowicz, A., et al., Fungal Genetics and Biology, 1999, 27, 175–185). Lignin is obtained as an aromatic compound through the process of de-polymerization of phenyl propanoid, a very complicated molecular unit, and becomes an important composition of cell wall of higher plant which provides power to resist outside attack.
Lignin peroxidase, Mn-dependent-peroxidase and laccase are the kind of lignin lyases.
As a polyphenol oxidase containing copper, laccase is called blue copper protein or blue copper oxidase and able to oxidize and resolve many toxic materials like phenol compound which is difficult to be resolved. With this resolving power, laccase effectively eliminates lignin from wood, which helps to enhance the quality of pulps.
Kraft process has been used to remove lignin from pulps. With this method about 90% of lignin can be extracted from pulps by cooking and by using NaOH, and the rest 10% of lignin is removed by using chlorine or chlorine dioxide. When chlorine compound is used to eliminate lignin, however, it co-responses with lignin to make chlorolignol, a chloro-organic compound, which is not only very slowly resolved in nature but also can cause problems in the immune and nervous systems of living matter. Thus, worldwide efforts have been made to legally prohibit against producing pollutants through lignin resolving process. The chemical resolving method of lignin has many problems economically and environmentally since this method consumes a lot of chemical compounds and requires lots of energy, and also produces many toxic pollutants containing chlorolignol (Boominathan and Reddy, Fungal Degration Lignin, 1992, 763–822).
When lignin is treated with lignin lyases, less energy and small amount of compounds are required and production of environmental pollutants could be reduced. Besides, lignin lyase is very important in the aspect of environmental protection since it exists in various types of natural systems and can resolve the aromatic compounds which are harmful for human and nature (Field, J. F., et al., Trend in Biotechnol., 1993, 11, 44–49).
Degradation-difficult organics such as saw dust, shavings, bark, waste ash, rice husk, rice straw, wheat straw, seed coat of beans, falling leaves, waste crop of oak mushrooms and other mushrooms are dejected in the field of agriculture and in other industries, and the carbon content ratio of those is 40–100%. Lignin takes 15–40% of their components, and lignin lyases resolve them effectively. Products after treated with lignin lyases can be used as organic fertilizer, which means industrial waste could be reused effectively.
Lignin lyases are also used in degradation of dye and decoloration of textile. More than 10,000 types of dyes are produced in the annual amount of 7×105 tons world widely from dyeing plant and dye-production plant. Synthetic dyes containing unresolving functional group such as azo group, nitro group and sulfon group are degradation-difficult toxic materials. Those still have been treated with physical, chemical methods which cost a lot and cause environmental contamination since there has been no other effective useful way so far. Thus, active studies have been made for the biological treatment of those degradation-difficult toxic materials using microorganism which is economical and has no need to worry about secondary contamination (Oh, K. G., et al., Kor. J. Appl. Microbiol. Biotechnol., 1999, 6, 500–508). There is a report concerning degradation of phenol-azo dye using laccase produced from Pyricularia oryzae(Chivukula, M. and V. Renganathan, Appl. Environ. Microbiol., 1995, 61, 4374–4377). And there is another report concerning degradation of aromatic compound using laccase produced from Trametes versicolor(Nicklas, Appl. Environ. Microbiol., 1989, 31, 70).
As of today, most of the reported microorganisms that produce lignin lyases are the fungus. The reported fungus are Agaricus bisporus belongs to white rot fungus, Pholiota aegerita, Podospora anserine, Trametes versicolor, Pleurotus ostreatus, and Phanerochaete chrysosporium, etc. (Chivukula, M. and V. Renganathan, Appl. Environ. Microbiol., 1995, 61, 4374–4377; Nicklas, Appl Environ. Microbiol., 1989, 31, 70; Perry, C. R., et al., J. General Microbiol., 1993, 139, 171–178; Rodriguez, C. S., et al., Biotech. Lett., 1997, 19, 995–998), and the reported bacteria are Burkholdera cepacia and Azospirillum lipoferum, etc. (Kinya, K. S., et al., Biotechno. Lett., 1998, 20, 459–462; Thurston, C. F., Microbiology, 1994, 140, 19–26).
The fungus such as white rot fungus grow slowly, and are not easily genetically mutated, so that it takes long time to culture and have difficulty in genetical uses. Meanwhile, bacteria are much useful sine they grow fast and are mutated easily. Degradation of lignin using fungus is too slow to use in industry because it can be induced only when carbon and nitrogen source are deficient. And fungus metabolize cellulose fiber as their first nutrient resulting in the reduction of the quality of pulp and yield of pulp production. Therefore, it is urgent and important to select a proper bacterium which can produce lignin lyases.
To overcome the foregoing and other disadvantages, we, the inventors of the present invention, have attempted to select a proper microorganism, a bacterium, producing lignin lyases which can resolve or treat degradation-difficult materials which cause many environmental problems. To select a proper microorganism, the present inventors have separated a microorganism from the gut of insect such as Sympetrum depressiusculm, and have confirmed that it belongs to Serratia family. And finally, the present invention is performed by verifying that this microorganism produces one or more lignin lyases among laccase, lignin peroxidase and Mn-dependent-peroxidase.